title: Identifying cellular mechanisms of zinc-induced relaxation in isolated cardiomyocytes.
abstract: We tested several molecular and cellular mechanisms of cardiomyocyte contraction-relaxation function that could account for the reduced systolic and enhanced diastolic function observed with exposure to extracellular Zn(2+). Contraction-relaxation function was monitored in isolated rat and mouse cardiomyocytes maintained at 37°C, stimulated at 2 or 6 Hz, and exposed to 32 μM Zn(2+) or vehicle. Intracellular Zn(2+) detected using FluoZin-3 rose to a concentration of ∼13 nM in 3-5 min. Peak sarcomere shortening was significantly reduced and diastolic sarcomere length was elongated after Zn(2+) exposure. Peak intracellular Ca(2+) detected by Fura-2FF was reduced after Zn(2+) exposure. However, the rate of cytosolic Ca(2+) decline reflecting sarcoplasmic reticulum (SR) Ca(2+)-ATPase (SERCA2a) activity and the rate of Na(+)/Ca(2+) exchanger activity evaluated by rapid Na(+)-induced Ca(2+) efflux were unchanged by Zn(2+) exposure. SR Ca(2+) load evaluated by rapid caffeine exposure was reduced by ∼50%, and L-type calcium channel inward current measured by whole cell patch clamp was reduced by ∼70% in cardiomyocytes exposed to Zn(2+). Furthermore, ryanodine receptor (RyR) S2808 and phospholamban (PLB) S16/T17 were markedly dephosphorylated after perfusing hearts with 50 μM Zn(2+). Maximum tension development and thin-filament Ca(2+) sensitivity in chemically skinned cardiac muscle strips were not affected by Zn(2+) exposure. These findings suggest that Zn(2+) suppresses cardiomyocyte systolic function and enhances relaxation function by lowering systolic and diastolic intracellular Ca(2+) concentrations due to a combination of competitive inhibition of Ca(2+) influx through the L-type calcium channel, reduction of SR Ca(2+) load resulting from phospholamban dephosphorylation, and lowered SR Ca(2+) leak via RyR dephosphorylation. The use of the low-Ca(2+)-affinity Fura-2FF likely prevented the detection of changes in diastolic Ca(2+) and SERCA2a function. Other strategies to detect diastolic Ca(2+) in the presence of Zn(2+) are essential for future work.
authors.affiliation: Department of Molecular Physiology and Biophysics, University of Vermont, Burlington, Vermont;
authors.forename: Ting
authors.initials: T
authors.lastname: Yi
authors.forename: Jonathan S
authors.initials: JS
authors.lastname: Vick
authors.forename: Marc J H
authors.initials: MJ
authors.lastname: Vecchio
authors.forename: Kelly J
authors.initials: KJ
authors.lastname: Begin
authors.forename: Stephen P
authors.initials: SP
authors.lastname: Bell
authors.forename: Rona J
authors.initials: RJ
authors.lastname: Delay
authors.forename: Bradley M
authors.initials: BM
authors.lastname: Palmer
mesh_terms: D000818:Animals; D002118:Calcium / Q000378:metabolism; D020746:Calcium Channels, L-Type / Q000187:drug effects / Q000502:physiology; D004305:Dose-Response Relationship, Drug; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D023421:Models, Animal; D009126:Muscle Relaxation / Q000187:drug effects* / Q000502:physiology*; D032383:Myocytes, Cardiac / Q000166:cytology / Q000187:drug effects* / Q000502:physiology*; D018408:Patch-Clamp Techniques; D051381:Rats; D011921:Rats, Inbred WKY; D017207:Rats, Sprague-Dawley; D019837:Ryanodine Receptor Calcium Release Channel / Q000378:metabolism; D012518:Sarcomeres / Q000187:drug effects; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases / Q000378:metabolism; D015032:Zinc / Q000494:pharmacology*
chemical_list: D020746:Calcium Channels, L-Type; D019837:Ryanodine Receptor Calcium Release Channel; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases; D015032:Zinc; D002118:Calcium
keywords: L-type channel; cardiac; myocyte; ryanodine receptor; sarcomere